SIRT5 functions as a tumor suppressor in renal cell carcinoma by reversing the Warburg effect

Background The aim of this study was to investigate the biological functions and underlying mechanisms of SIRT5 in clear cell renal cell carcinoma (ccRCC). Methods SIRT5 expression data in The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) were selected, and the correlations between SIRT5 expression and various clinicopathological parameters were analysed. SIRT5 expression in ccRCC tissues was examined using immunohistochemistry. Stable cell lines with SIRT5 knockdown were established. In vitro and in vivo experiments were conducted to investigate the functional roles of SIRT5 in the cellular biology of ccRCC, including cell viability assays, wound healing assays, soft agar colony formation assays, Transwell invasion assays, qRT–PCR, and Western blotting. In addition, microarrays, rescue experiments and Western blotting were used to investigate the molecular mechanisms underlying SIRT5 functions. Results SIRT5 expression was downregulated in ccRCC compared with normal tissues, which correlated with a poor prognosis of ccRCC. SIRT5 knockdown significantly increased cell proliferation, migration and invasion in vitro. In vivo experiments revealed that SIRT5 knockdown promoted ccRCC tumorigenesis and metastasis. Mechanistically, SIRT5 deglycosylated PDHA1 at K351 and increased PDC activity, thereby altering the metabolic crosstalk with the TCA cycle and inhibiting the Warburg effect. SIRT5 overexpression was related to low succinylation of PDHA1. Conclusions Downregulated SIRT5 expression in ccRCC accelerated the Warburg effect through PDHA1 hypersuccinylation and induced tumorigenesis and progression, indicating that SIRT5 may become a potential target for ccRCC therapy.

SIRT3 deacetylates PDHA1 at K321, recruits PDP1 and finally increases PDC activity [21,22]. Another mitochondrial SIRT4 hydrolyses the lipoamide cofactor from the E2 component dihydrolipoyllysine acetyltransferase (DLAT) and reduces PDH activity [23]. Recently, SIRT5, one of the three mitochondrial sirtuins, was reported to desuccinylate various proteins, such as PDHA1 and SHDB, and regulate the activities of these proteins [20,24]. However, the relationship between SIRT5 and cancer metabolism in ccRCC remains elusive. In this study, we aimed to investigate the expression and potential role of SIRT5 in ccRCC.

Sample collection from patients with ccRCC
This study was approved by the Ethics Committee of Shanghai Ruijin Hospital, and all patients provided written informed consent. ccRCC specimens (n = 280) that were histopathologically confirmed by three independent pathologists were prepared as a tissue microarray with matched normal sections. Six paired tumor and adjacent normal tissues were also obtained from patients with ccRCC who underwent radical nephrectomy in our institution. Clinical information was obtained from the medical records, including age, sex, tumor size, grade, and stage. Tumor samples and adjacent normal tissues were placed in liquid nitrogen followed by storage at −80 °C. In addition, computed tomography (CT)-guided biopsies were collected from two patients with ccRCC, and patient-derived organoids (PDOs) were established and cultured.  (lacking the mitochondrial signal peptide,  1-36 AAs), wild-type PDHA1, and PDHA1 mutants were cloned into the pcDNA3.1-Flag, pcDNA3.1-Myc and pcDNA3.1-HA vectors, respectively. The SIRT5specific shRNA (5'-GCT GGA GGT TAT TGG AGA A-3' , 5'-CAG CAT CCC AGT TGA GAA A-3') was used to knock down SIRT5 expression, and the nonsilencing shRNA oligonucleotide was used as a negative control. The anti-pan-succinyl lysine antibody and anti-succ-K351 of PDHA1 antibody were generated in our lab. Synthetic PDHA1 peptides containing succinylated K351 were used as antigens to immunize rabbits, and purified serum proteins were measured. Antibodies specific to pyruvate dehydrogenase E1-alpha subunit antibody (Abcam), SIRT5 (Sigma), β-actin (Genscript), Flag-Tag (Abmart), Myc-Tag (Abmart), HA-Tag (Cell Signaling Technology), and mitochondria (Abcam) were purchased. PDH activity was determined using a PDH Enzyme Microplate Assay Kit (Abcam ab109902).

In vitro succinylation and desuccinylation
We succinylated the lysine-containing peptides (GL Biochem) by mixing 0.5 mM succinyl-CoA (Sigma) and 50 ng/μl peptides in 30 mM HEPES (pH 7.4) in a 15 μl system and incubating them at 37 ℃ for 3 h. The peptides were desalted using C18 ZipTips (Millipore) prior to the MALDI-TOF MS analysis. Desuccinylation was carried out by incubating enzymes with SIRT5 (Sigma) at 37 °C for 3 h in 30 mM HEPES buffer (6 mM MgCl 2 , 1 mM DTT, and 1 mM NAD + , pH 7.4). An in vivo lung metastasis model was generated using male nude BALB/c mice aged 4 weeks by a tail vein injection of Caki-1 cells stably expressing luciferase. Lung metastatic progression was monitored using the IVIS-100 system (Caliper Life Sciences). In vivo BLI was performed every 7 days, and Living Image ® software was used to analyse the data. Mice were euthanized 28 days after implantation, and lung metastatic lesions were analysed using the BLI system.

Western blot analysis
Western blot analyses were performed using standard procedures. After cells were harvested, the protein concentration was determined using Quantity One software (Bio-Rad, Hercules, Calif., USA). Cell lysates were separated on 10% SDS-PAGEPAGE gels, transferred to PVDF membranes (Millipore, Bedford, MA, USA) and blocked with 5% w/v skim milk for 2 h at room temperature. Anti-PDHA1, anti-Succ-K351 PDHA1, anti-SIRT5, anti-GAPDH, and anti-actin antibodies were incubated with the membrane overnight at 4 °C. Immune complexes were detected with a horseradish peroxidase-conjugated secondary antibody (1:3000, Southern Biotech) for 2 h at RT. Finally, bands were visualized using an enhanced chemiluminescence system (ECL; Pierce Company Woburn, MA, USA). Western blot signals were obtained by detecting chemiluminescence with a Typhoon fla9500 system (GE Healthcare). ImageJ software (NIH) was used to analyse the densities of the bands. Each blot shown in the figures is representative of at least three experiments.

Immunofluorescence analysis
Standard procedures were used for the immunofluorescence analysis. Cells were seeded in 24-well plates, fixed with 4% paraformaldehyde, and then permeabilized with 1% Triton. Cells were incubated overnight at 4 °C with anti-PDHA1 and SIRT5 antibodies and detected the next day with an Alexa Fluor 555-conjugated goat anti-mouse IgG antibody. The nuclei were stained with DAPI (Sigma). Immunofluorescence images were observed with a fluorescence microscope (Leica, DMI4000B).

Measurement of the activity of PDHA1 complex
PDHA1 activity was measured in a reaction buffer containing 50 mM KH 2 PO 4 (pH 7.0), 1 mM MgCl 2 , 2 mM sodium pyruvate, 0.2 mM thiamine diphosphate and 0.1 mM 2,6-dichlorophenolindophenol (2,6-DCPIP). The purified PDHA1/PDHB complex was added to start the reaction. The reaction was maintained at 30 °C. The course of the reaction was monitored by measuring the reduction of 2,6-DCPIP at 600 nm using a Roche spectrophotometer.

Oxygen consumption rate (OCR)
KMRC-20 cells or Caki-1 cells were seeded on XFe24 cell culture microplates (Seahorse Biosciences) at densities of 40,000 or 15,000 cells/well, respectively. The analysis was performed using the XF cell Mito stress test kit (Seahorse Bioscience) according to the manufacturer's protocol. The culture medium was replaced with assay medium (XF Base Medium containing 5.5 mM glucose, 2 mM glutamine, 1% FBS, 1 nM insulin, and 100 nM dexamethasone, pH 7.4) (Seahorse Bioscience) 1 h before the analysis. Oligomycin, FCCP, rotenone and antimycin A used in assays were at final concentrations of 2 μM, 1 μM, 1 μM and 1 μM, respectively. The results were normalized to the cell numbers.

Measurement of the growth curves
PDHA1 knockout cells were transfected with SIRT5, PDHA1, PDHA1 + SIRT5, PDHA1 K351Q or PDHA1 K351Q + SIRT5 and seeded in 96-well plates. Cell morphology was observed under an inverted microscope, the plate and its contents were allowed to equilibrate at room temperature for approximately 30 min, the transparent bottom was affixed with a white back cover, and the luminescence was recorded with EnSpire.

Soft agar colony formation assay
For soft agar colony formation assays, 2 ml of medium containing 10% FBS and 0.7% agar were used. Cells were seeded in 2 ml of medium containing 10% FBS with 0.35% agar at a density of 1 × 10 5 cells per well and incubated for 21 days at 37 °C. Then, the number of colonies that formed in soft agar was counted using ImageJ software, and images were captured using an Olympus IX5 microscope.

Wound-healing assay
Cell migration was assessed by performing a wound healing assay. Briefly, Caki-1 cells were transfected with PDHA1, PDHA1 + SIRT5, PDHA1 K351Q or PDHA1 K351Q + SIRT5. Approximately 5 × 10 6 cells were seeded into 24-well plates and cultured for 24 h. Then, a yellow plastic pipette tip was used to create a wound by scraping the cells. Cell migration was monitored under a Nicon Eclipse microscope and photographed at 100× .

Transwell invasion assay
Cell invasion experiments were carried out using 24-well Transwell plates with 8 μm pore size polycarbonate Matrigel-coated membrane inserts according to the manufacturer's instructions. After 20 h of incubation, noninvading cells in the upper insert were removed, and cells that had invaded into the lower Matrigel surface were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Ten random fields of view were quantified.

Immunohistochemistry
Formalin-fixed and paraffin-embedded specimens were prepared for histological sectioning. Antigen recovery was performed on renal carcinoma specimens incubated with Tris-EDTA buffer (pH 8.4) at 99 °C for 60 min. Endogenous peroxidase activity was inactivated by incubating sections with a methanol and 3% H 2 O 2 solution. Sections were incubated with the primary antibody for 60 min, secondary antibody for 8 min, and DAB developer for 8 min. All procedures were performed using the Ventana BenchMark XT automated stainer, and the sections were scanned using a Ventana iScanCoreo scanner. IHC results were quantified by experienced pathologists. The intensity was calculated according to the positive area and the degree of positive staining. The sections were stained with SIRT5 (1:100), PDHA1 (1:100), Succ-K351-PDHA1 (1:100) and Ki67 (1:100) antibodies using an ultraView Detection Kit.

Statistical analysis
Data are presented as the means ± standard deviations (SD), and bars in the graphs represent standard deviations. Statistical analyses were conducted using SPSS 22.0 software (IBM, Corp., Armonk, NY, USA). Significant differences between groups were determined using Student's t test. The significance level for statistical testing was set to P < 0.05.

SIRT5 is downregulated and associated with a poor prognosis for patients with ccRCC
The RNA-seq datasets and clinical information of patients with ccRCC were downloaded from The Cancer Genome Atlas (TCGA; https:// portal. gdc. cancer. gov/). We performed a comprehensive bioinformatics analysis and found significantly lower SIRT5 expression in ccRCC tissues than in normal tissues (P = 8.308e −16 ) (Fig. 1A).
We further detected the expression of SIRT5 in 72 paired ccRCC tumor samples obtained from Ruijin Hospital and found that SIRT5 expression was significantly decreased in the tumor samples compared with paracancerous normal tissues (P = 4.117e−09) (Fig. 1B). In addition, patients with ccRCC in TCGA-KIRC cohort with lower SIRT5 expression levels had a higher clinicopathological TNM stage and grade ( Fig. 1C-G). The Kaplan-Meier analysis verified that patients with lower SIRT5 levels had a worse overall survival prognosis (Fig. 1H). We further performed a weighted gene coexpression network analysis (WGCNA) based on TGCA-KIRC samples and found that the correlation coefficient between SIRT5 levels and identified MEred modules was 0.236 (P = 0.0002) with a threshold of 12 ( Fig. 1I-K). The GO functional enrichment analysis indicated that SIRT5 was primarily related to mitochondrial respiratory chain complex assembly and MAP2K/MAPK activation (Fig. 1L). The KEGG pathway analysis also indicated that SIRT5 participated in the fatty acid metabolism pathway and propionic acid metabolism pathway (Fig. 1M). Based on these data, we concluded that SIRT5 might be a tumor suppressor that is involved in ccRCC tumorigenesis by regulating cancer metabolism.

SIRT5 desuccinylates PDHA1
Wild-type SIRT5 decreased the pansuccinylation of PDHA1 ( Fig. 2A), suggesting that PDC activity might be regulated by SIRT5. We tested this hypothesis by first analysing the interaction between SIRT5 and PDHA1. SIRT5 was copurified with PDHA1, and PDHA1 was copurified with SIRT5 when they were coexpressed in HEK293T cells (Fig. 2B and C), suggesting that SIRT5 and PDHA1 interacted with each other. Incubation of recombinant PDHA1 (rPDHA1) with succinyl-CoA resulted in a gradual increase in the levels of succinylated PDHA1 in a succinyl-CoA-dependent manner (Fig. 2D), confirming that PDHA1 was succinylated directly. Moreover, SIRT5 overexpression removed the succinylation of PDHA1 in the cell line stably expressing SIRT5 (Fig. 2E), whereas SIRT5 knockdown increased PDHA1 succinylation

SIRT5 desuccinylates K351 of PDHA1
We determined which lysine sites of PDHA1 were desuccinylated by SIRT5 by mutating each candidate lysine site of PDHA1 according to previously published data [15,18]. SIRT5 had no effect on the succinylation of K77, K121 and K374 in PDHA1 but desuccinylated K351 in PDHA1 (Fig. 3A), suggesting that K351 in PDHA1 might be the substrate of SIRT5. Interestingly, SIRT5 only decreased the succinylation of wild-type PDHA1 but not its desuccinylation mimetic K351R or succinylation mimetic K351Q mutants, indicating that K351 might be the substrate of SIRT5 (Fig. 3C). We verified that K351 in PDHA1 is the substrate of SIRT5 by generating site-specific antibodies against Succ-K351 of PDHA1 using synthetic succinylated PDHA1 peptides as antigens (Fig. 3B).
The levels of Succ-K351 in endogenous PDHA1 were measured following SIRT5 overexpression and knockdown to test whether SIRT5 desuccinylated endogenous PDHA1. PDHA1 was knocked down and then reintroduced into the HEK293 cell line to exclude a potential effect of endogenous PDHA1. SIRT5 decreased the K351 succinylation of wild-type PDHA1 but had no effect on the K351 mutants (Fig. 3D). Overexpression of wild-type SIRT5 in HeLa cells decreased the levels of endogenous Succ-K351 PDHA1; however, overexpression of the mitochondrial localization defective but functional mutant (SIRT5 ∆50 ) did not (Fig. 3E). Conversely, SIRT5 knockdown increased the level of Succ-K351 in endogenous PDHA1 (Fig. 3F). These results collectively supported the hypothesis that K351 in PDHA1 might be the site desuccinylated by SIRT5.

SIRT5 desuccinylates PDHA1 and regulates metabolic pathways
We confirmed that SIRT5 desuccinylates PDHA1 and reroutes the metabolic pathways by first testing the effect of succinyl-CoA on the succinylation and activity of PDHA1. Succinyl-CoA increased the succinylation of PDHA1 but decreased its activity (Fig. 4A). Then, the succinylation and activity of PHDA1 were evaluated under conditions of SIRT5 overexpression or knockdown were evaluated. Overexpression of SIRT5 decreased PDHA1 succinylation but increased its activity (Fig. 4B). C Lysine 351 (K351) of PDHA1 was mutated to arginine (R) or glutamine (Q) and then transfected into HEK293T cells with or without SIRT5, and the pansuccinylation level of PDHA1 was determined. D K351R and K351Q PDHA1 mutants were transfected into HEK293T cells with or without SIRT5, and the succinylation level of PDHA1 was determined. E SIRT5 ∆50 was transfected into HeLa cells, and the succinylation level of endogenous PDHA1 was determined. F The succinylation levels of endogenous PDHA1 in HeLa cells before and after SIRT5 knockdown by independent shRNAs were compared Conversely, SIRT5 knockdown increased PDHA1 succinylation and decreased its activity (Fig. 4C). We further evaluated the OCR and metabolites of glycolysis and the TCA cycle to confirm that metabolic  19:521 suggesting that SIRT5 regulated the OCR of KMRC-20 cells. Additionally, SIRT5 knockdown decreased the OCR of Caki-1 cells (Fig. 4E). Moreover, we analysed the metabolites of glycolysis and the TCA cycle and found that SIRT5 decreased the concentrations of glucose-6-phosphate (G6P), 3-phosphoglyceric acid (3-PG), pyruvate and lactate and increased citrate levels (Fig. 4F), indicating that SIRT5 decreased glycolysis and increased the TCA cycle. Last, SIRT5 overexpression dramatically decelerated Caki-1 proliferation, whereas SIRT5 knockdown significantly promoted Caki-1 proliferation (Fig. 5A). PHDA1 knockdown in Caki-1 cells increased proliferation, but when both PDHA1 and SIRT5 were reintroduced, cell proliferation decreased significantly (Fig. 5B). Interestingly, overexpression of PDHA1 K351Q increased the proliferation of Caki-1 cells, but SIRT5 had no effect on this cell line. SIRT5 knockdown also significantly promoted proliferation ( Fig. 5C and D) and migration (Fig. 5E and G), whereas SIRT5 inhibited cell migration through PDHA1 but did not reverse the PDHA1 K351Q -mediated increase in cell migration (Fig. 5F). At the same time, ccRCC organoid models were established to confirm the potential clinical value of SIRT5. SIRT5 knockdown significantly increased cell proliferation in the PDOs generated from two different patients with ccRCC (Fig. 5H). These results supported the hypothesis that desuccinylation of PDHA1 by SIRT5 regulated metabolic pathways and inhibited cell proliferation, migration and invasion of ccRCC.

SIRT5 correlates with PDHA1 hyposuccinylation and progression of ccRCC
Immunohistochemistry and Western blot assays were conducted using 6 paired ccRCC specimens to illustrate the differential expression of each marker in normal and tumor tissues. SIRT5 was expressed at low levels in tumor tissues, and levels of PHDA1 succinylated at K351 were observed in ccRCC samples ( Fig. 6A and B). The results provided evidence that hypersuccinylation of PDHA1 promoted ccRCC progression, which was reversed by overexpression of SIRT5. Immunohistochemical staining and statistical analysis of the Ruijin-ccRCC cohort (n = 280) showed that SIRT5 expression was significantly decreased in ccRCC samples compared with normal tissues (Fig. 6C). The progression-free survival rate of patients with low SIRT5 expression was significantly higher than that of patients with high SIRT5 expression (P < 0.05) (Fig. 6D). The animal study showed that the tumor size of the SIRT5-KD group was significantly larger than that of the control group (Fig. 6E). HE staining and immunohistochemistry also showed that the percentages of Ki-67-positive cells were apparently increased in the SIRT5-KD group (Fig. 6F). SIRT5 knockdown significantly promoted metastatic processes compared with the control group in a lung metastasis model (Fig. 6G). Based on these results, SIRT5 suppressed ccRCC tumorigenesis by regulating the hyposuccinylation of PHDA1.
In the current study, we found that SIRT5 was significantly downregulated in ccRCC tissues compared with normal tissues and associated with a poor prognosis. SIRT5 deficiency significantly facilitated cell proliferation, migration, and invasion in vitro and promoted ccRCC tumorigenesis and metastasis in vivo. SIRT5 overexpression significantly decreased the oxygen consumption rate and inhibited cell proliferation and migration in ccRCC. The underlying mechanism showed that downregulated expression of SIRT5 in ccRCC resulted in hypersuccinylated PDHA1 at K351 and decreased PDC activity, thus accelerating the Warburg effect (Fig. 7). The results of our study showed that SIRT5 functioned as a tumor suppressor in ccRCC and correlated with metabolic reprogramming during tumor growth and metastasis by regulating the hyposuccinylation of PHDA1.
SIRT5 has been previously reported as a master metabolic regulator, and its expression levels are significantly associated with the metabolic patterns and functions of various cell types [34]. SIRT5 not only maintains mitochondrial function through posttranslational modifications of mitochondrial proteins and enzymes but also modulates different pathways, including glucose oxidation, ketone body formation, fatty acid degradation, ammonia disposal, and redox homeostasis. Furthermore, SIRT5 appears to be a key determinant of metabolic rewiring in response to environmental stress [35].
Despite these impactful findings, our study has some limitations. First, we have not yet clearly determined how to define high and low expression of SIRT5, and further immunohistochemical studies in a large cohort are necessary to identify the optimal cut-off value. Second, since isocitrate dehydrogenase 2 and glucose 6-phosphate dehydrogenase, which are electron transport chain complexes, are regulated by SIRT5, a meaningful study would be to determine whether SIRT5 regulates metabolic reprogramming by targeting other related enzymes. Moreover, a systematic description of whether SIRT5 is associated with other clinical phenotypes, such as stem cells, angiogenesis, and tyrosine kinase inhibitor resistance, is needed. Future research based on a more comprehensive study design is needed to explore the underlying mechanism.

Conclusions
Downregulated expression of SIRT5 in ccRCC accelerated the Warburg effect through PDHA1 hypersuccinylation and finally caused tumorigenesis and progression, which were reversed by SIRT5 overexpression. Our study suggested that SIRT5 may function as a potential target for ccRCC therapy.